Fuel ethanol produced from renewable resources is one of the long-term solutions to global fossil fuel shortages, rising energy costs, and global warming effects related to increased atmospheric carbon dioxide. Fuel ethanol from renewable resources is produced by fermentation of sugars using a biocatalyst. Currently yeast is the biocatalyst most widely used for ethanol production. Fermentable sugars are most typically obtained from processed biomaterials including corn grain, sugar beets, and sugar cane. An alternative abundant biomaterial sugar source is cellulosic or lignocellulosic biomass. Methods are being developed for processing of cellulosic and lignocellulosic biomass to produce fermentable sugars using physical, chemical, and/or enzymatic treatments.
It is difficult to maintain sterility in a large scale fermentation process, particularly when biomaterial is used as as a carbohydrate source. Large scale fermentation processes are typically contaminated with bacteria that may come from the processed biomaterial, equipment, process water or other sources. Typically contaminating bacteria are lactic acid bacteria (LAB) such as Lactobacillus species. Contaminating bacteria reduce fermentation product yield by utilizing sugars and reducing effectiveness of the primary product biocatalyst. Contaminating bacteria produce undesired products such as acetic and lactic acid which increase stress conditions in a culture leading to poorer growth of the biocatalyst and/or lower production of the biocatalyst product.
Contaminating bacteria, predominantly lactic acid bacteria, have been a problem in fermentations that use yeast as the biocatalyst, typically with mash or molasses used as the carbohydrate source for ethanol production for either fuel or brewing. Due to differential sensitivities of yeast and contaminating bacteria to some antimicrobials, a number of antimicrobials can be used to control bacteria in yeast fermentations. Antimicrobials successfully used in yeast fermentations to control LAB contamination include penicillin (Day et al. (1954) Agricultural and Food Chemistry 2:252-258), virginiamycin (Hynes et al. (1997) J. of Industrial Microbiology & Biotechnology 18:284-291; Bischoff et al. (2009) Biotechnology and Bioengineering 103:117-122; WO2007145857), hop acids (US20090042276), FermaSure™, as well as erythromycin, tylosin, and tetracycline.
Zymomonas is being developed as an effective biocatalyst for producing ethanol by engineering strain improvements including utilization of xylose and arabinose in addition to glucose, and inactivating competing metabolic pathways. In addition, Zymomonas has been adapted for use in hydrolysate fermentation medium by increasing tolerance to inhibitors present in cellulosic biomass hydrolysate. However, using Zymomonas as a biocatalyst for ethanol fermentation presents additional challenges in contamination control since this biocatalyst is a bacterium, as are the predominant contaminants.
Concentrations of many antibiotics that are safe to use with yeast are inhibitory to growth of Zymomonas mobilis strain ZM4, including tetracycline, kanamycin, polymixin and streptomycin (Agrawan and Basappa, Biotechnology Letters (1996) 18:673-678). Only penicillin G was shown to be safe for use with Zymomonas. Benzyl penicillin was successfully used to control bacterial contamination in batch Zymomonas mobilis fermentation for ethanol production (Grote and Rogers, Journal of Fermentation Technology (1985) 63:287-290). In another review Zymomonas was reported as a contaminant of cider and beer, and strains of Zymomonas with resistance to typically used levels of some antibiotics including kanamycin, polymyxin, and sreptomycin were found (Swings and De Ley, Bacteriological Reviews (1977) 41:1-46). Differences among strains may be related to the encoding of resistance on plasmids, as was found for streptomycin, kanamycin, and gentamicin in Z. mobilis strain CP4 (Walia et al. (1984) Applied and Environmental Microbiology 47:198-200).
There remains a need for methods to control bacterial contaminants in fermentations that use a bacterial Zymomonas biocatalyst that has been developed for ethanol production.